University of Vermont ScholarWorks @ UVM UVM Honors College Senior Theses Undergraduate Theses 2015 Inhibitory Effects of Ethanol on the NLRP3 Inflammasome Laura Rose Hoyt The University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/hcoltheses Recommended Citation Hoyt, Laura Rose, "Inhibitory Effects of Ethanol on the NLRP3 Inflammasome" (2015). UVM Honors College Senior Theses. 194. https://scholarworks.uvm.edu/hcoltheses/194 This Honors College Thesis is brought to you for free and open access by the Undergraduate Theses at ScholarWorks @ UVM. It has been accepted for inclusion in UVM Honors College Senior Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. Inhibitory Effects of Ethanol on the NLRP3 Inflammasome An Honors Thesis Presented By Laura Hoyt To The College of Arts and Sciences The University of Vermont Advised by Matthew E. Poynter, PhD In Partial Fulfillment of the Requirements for College Honors in the College of Arts and Sciences November 2015 1 Abstract Immunosuppression is a major complication of alcoholism and contributes to increased rates of opportunistic infections and sepsis associated with the addiction. The NLRP3 inflammasome is a central intracellular pattern recognition receptor within the innate immune system, which leads to the cleavage and secretion of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18. Ethanol has been reported to inhibit IL-1β secretion, and here we verify that the alcohol can specifically inhibit activation of the NLRP3 inflammasome resulting in attenuated IL-1β and caspase-1 cleavage and secretion, as well as ASC secretion in response to several agonists. These results were found to be independent of the activation of GABAA receptors or the inhibition of NMDA receptors. Ethanol was only partially able to prevent IL-1β secretion subsequent to NLRC4 activation and was incapable of preventing NLRP1b dependent IL-1β secretion, which are both largely independent of the adapter protein ASC, and ethanol was shown to prevent the formation of ASC specks. Treatment of cells with ethanol resulted in markedly decreased global tyrosine phosphorylation, while administration of the tyrosine phosphatase inhibitor sodium orthovanadate prior to ethanol restored IL-1β secretion. Multiple alcohol containing organic compounds exerted inhibitory effects on the NLRP3 inflammasome parallel to ethanol; however, isoamyl alcohol’s non-alcohol analog, 2-methylbutane, did not. Together, these results show that ethanol antagonizes the NLRP3 inflammasome at an apical event in its activation potentially through the stimulation of protein tyrosine phosphatases. As other short chain alcohols retain this ability, this effect could be dependent on the hydroxyl group of these compounds. 2 Introduction Inflammasomes are a family of large multi-protein intracellular pattern recognition receptors (PRRs) that respond to a wide variety of exogenous pathogen associated molecular patterns (PAMPs) and endogenous danger associated molecular patterns (DAMPs), facilitating the secretion of the pro-inflammatory cytokines, IL-1β and IL-18, as well as a form of inflammatory cell death known as pyroptosis (1). Unlike many innate immune pathways, stimulation of a functional inflammasome requires two steps. During priming (step 1), activation of the transcription factor NF-κB, downstream of the stimulation of many PRRs, leads to the production of several components of the inflammasome and the secretion of the pro- inflammatory cytokine TNFα (2). Activation of the inflammasome (step 2) requires the exposure of cells to a separate set of PAMPs and DAMPs, which work through unique signaling pathways leading to the oligomerization of one of several different Nucleotide Oligomerization Domain (NOD)-Like Receptor (NLR) proteins, the adaptor protein Apoptosis-associated Speck-like protein containing a CARD (ASC), and pro-caspase-1 into an organized inflammasome complex (3). This oligomerization is mediated by homotypic PYRIN-PYRIN domain binding between NLRs and ASC, and CARD-CARD interactions between ASC and pro-caspase-1, resulting in the formation of a discrete ASC speck within stimulated cells (4). These ASC specks form rapidly and irreversibly within activated cells and are a platform for efficient pro-IL-1β and pro- IL-18 cleavage. While the activity of all inflammasomes is thought to be enhanced by the incorporation of ASC into their complexes, NLRP1 and NLRC4 contain their own CARD domains and can interact directly with pro-caspase-1 independent of ASC (5). This assembly allows for the conversion of pro-caspase-1 into an active caspase-1 enzyme, which cleaves pro- IL-1β and pro-IL-18 into their mature, secreted forms. These cytokines then function to promote 3 vasodilation, attract and stimulate neutrophils, induce fever, and activate the acute phase response within an organism (6). Some consider the secretion of IL-1β and IL-18 to be a third step in the process of inflammasome activation. Both IL-1β and IL-18 are leaderless proteins, which despite years of research and many proposed models, still do not have a well-defined mode of release (7). The final outcome of inflammasome formation, pyroptotic cell death, is believed to amplify the immune response while depleting pathogens of their host leukocyte niche (8). The NLRP3 inflammasome is capable of responding to a particularly diverse set of PAMPs and DAMPs, including ATP, nigericin, alum, asbestos, silica, and cholesterol crystals (9-13). These agonists activate the inflammasome through disparate pathways, such as K+ efflux and lysosomal rupture, eventually converging on ASC phosphorylation and multimerization (14, 15). As a result, this inflammasome, expressed predominantly by macrophages, but also monocytes, neutrophils, dendritic cells, some lymphocytes, and cells that are not leukocytes, plays a major function in immune homeostasis (16). Beyond its protective roles in response to pathogens, over-activation of the NLRP3 inflammasome has been implicated in the pathogenesis of an array of diseases such as atherosclerosis, diabetes, gout, and multiple sclerosis (17-19). Similarly, gain of function mutations in NLRP3 lead to the set of debilitating diseases known as Cryopyrin-Associated Periodic Syndrome (CAPS) (20). Although many inhibitors of signal 1 are known, until recently few compounds capable of directly inhibiting signal 2 were discovered. Alcohol use disorders were estimated to be the third most common non-genetic cause of mortality in the U.S. in the year 2000 (21). Alcohol abuse predisposes individuals to opportunistic infections and organ damage, which are the two most prominent alcohol-related medical complications (21). In trauma and post-surgical patients, alcohol exposure occurring 4 prior to or at the time of injury enhances morbidity and mortality due to increased rates of sepsis and shock, and chronic alcoholics account for 50% of all Acute Respiratory Distress Syndrome patients (22, 23). Furthermore, light to moderate alcohol consumption is associated with decreased risks of developing coronary artery disease and atherosclerosis, illnesses commonly associated with systemic inflammation (17). The pattern of drinking differentially affects the consequences of alcohol abuse. Binge alcohol consumption suppresses host innate immune defense, while chronic alcohol consumption suppresses innate and adaptive immune systems, yet activates chronic inflammation (24). Ethanol is a known inhibitor of signal 1 (the consequences of PRR signaling) and its consumption is associated with decreased circulating levels of TNFα and IL-1β (25, 26). Recently, ethanol, but not its metabolite acetaldehyde, was found to also be capable of inhibiting signal 2 for the NLRP3 and AIM2 inflammasomes (17). The methods through which ethanol exerts its immunosuppressive effects are still unclear, yet given the central role that inflammasomes play in the immune response, it is possible that direct inhibition of signal 2 could be an important target of alcohol induced immunosuppression. Ethanol is known to have a wide range of effects when administered to cells. At high doses, it can alter membrane fluidity and can diffuse across the plasma membrane to interact with cytosolic proteins (27). Some known intracellular effects of acute ethanol administration include tyrosine phosphatase and adenylyl cyclase activation (28, 29). At lower doses, ethanol is thought to interact with a variety of cell surface receptors, particularly neurotransmitter receptors, in an agonistic or antagonistic manner (30, 31). During chronic exposure to ethanol, gene expression can be altered, potentially contributing to the differences that chronic alcoholism and binge drinking exert on immune function (32). 5 The goal of this study was to further elucidate the mechanism of ethanol’s inhibition of the NLRP3 inflammasome, primarily using the J774 mouse macrophage and THP-1 human monocyte cell lines and a protocol resembling binge drinking in humans. Experiments were designed to assess ethanol’s ability to directly inhibit signal 2, rather than its already well- defined capacity to prevent NF-κB and signal 1 activation. By identifying pathways involved in ethanol’s blockade of this key innate immune complex, we hope to better understand and determine potential sites of therapeutic intervention in ethanol mediated immunosuppression and also to identify potential targets for future NLRP3 inflammasome inhibitors.